1. Field of the Invention
The present invention relates to components, methods, and analytical devices for fast point of care or field colorimetric analysis. More specifically, the invention relates to the use of nanoparticles as active color agents to fabricate colorimetric assays for the detection of substances and to their practical applications in a variety of fields including clinical diagnosis, environmental and food.
2. Description of the Related Art
The invention consists of a colorimetric assay (including a method, test device, test strip, detection kit, biosensor) for the visual analysis of chemical substances in various samples. The method incorporates a new concept based on a novel colorimetric component. The colorimetric component refers to nanoparticles of cerium oxide, or ceria, which change the color in response to the presence of a particular analyte, and of cerium oxide nanoparticles in close contact with specific oxidase enzymes, in response to a substrate or product of the enzymatic reaction. The nanoparticles can be used in solution, or attached to a solid support to construct a device. The device is fabricated by immobilizing cerium oxide nanoparticles with and without enzymes onto a solid support. Examples of suitable solid supports include but are not limited to paper, ceramics, membrane, packaging materials, polymeric support, cotton swab, patch, test tube, wipe, gas or fluid collection device, sponge, and lens. The device can be used to determine quantitatively the presence and the relative concentration of chemicals including, but not limited to, hydrogen peroxide, reactive oxygen species and free radicals, ethanol, glucose, cholesterol, billirubin, glutamate, lactate, and various antioxidants.
Hydrogen peroxide is a key component in many chemical, biological, pharmaceutical, clinical, environmental and food processes. The availability of a reliable, efficient and economic method for its detection is of great practical importance. For example, the use of hydrogen peroxide has become a popular method of water treatment because peroxide is effective in elimination of bacteria and moulds. Cheap and effective monitoring methods of hydrogen peroxide in pools, hot tubs and drinking water are needed. Monitoring peroxide concentrations is also important in hospitals where hydrogen peroxide is used as a sterilizing and cleaning solution. Because hydrogen peroxide is prone to decomposition, losing its antibacterial potency overtime, periodic confirmation of the concentration of these solutions is necessary. At the same time, hydrogen peroxide is an indicator of inflammation and it is known that wound-induced extracellular H2O2 may reach concentrations of 0.5-50 mM near the wound area. Fast detection kits of hydrogen peroxide could also find potential uses for screening of hydrogen peroxide-generating bacteria, (e.g. lactic acid bacteria), present in healthy human micro flora and in food industry (e.g. milk products, sausage production). In addition, hydrogen peroxide is the by-product of many enzymatic reactions of oxidase enzymes (e.g. glucose oxidase, lactate oxidase, glutamate oxidase, cholesterol oxidase, alcohol oxidase, etc) and its detection represents the basis of numerous enzyme biosensors e.g. for quantitative determination of glucose, lactate, glutamate, cholesterol, ethanol, etc). Quantitative analysis of hydrogen peroxide as well as oxidase enzyme substrates listed above has been accomplished using electrochemical methods based on direct reduction or oxidation of H2O2 at the surface of a chemically or enzymatically modified working electrode. Such systems are used in conjunction with reference/counter electrodes and a potentiostat.
Several colorimetric test strip devices for these substances have been reported. These devices use colorimetric reagents (e.g. soluble dyes), fluorescent compounds, redox reagents and enzymes, to quantitatively determine specific substances in a sample. In previous reports, an oxidase enzyme (e.g. glucose oxidase, cholesterol oxidase, alcohol oxidase, etc), in solution or immobilized onto a solid surface constituting the sensing area, is used to catalytically transform the enzyme substrate (glucose, cholesterol, ethanol, etc) to hydrogen peroxide. The enzymatically generated hydrogen peroxide is subsequently measured using chromogenic substances and spectrophotometric analysis. The response of the sensor is based on a color change of a dye, added in solution, in response to a chemical and/or enzymatic reaction. The intensity of the color is typically compared to that of several standard color charts obtained with known concentrations of analyte. In many cases these test strips involve the use of multiple compartments and separate reagents (chromogens for the color change, enzymes, co-substrates, etc) that need to be added in order to initiate the desired colored reaction. In most cases, ABTS (2, 2′-azino-di(3-ethylbenzthiazoline-6-sulfonate) is used as a chromogenic compound for the detection of hydrogen peroxide. In previously developed paper based enzymatic assays, the soluble dye (e.g. KI) migrates to the sensing spot by capillary action. In the new colorimetric assay described here, redox active ceria nanoparticles are used as a chromogenic indicator for H2O2, eliminating the need for both the organic dye and the peroxidase enzyme.
Traditionally, cerium oxide or ceria, or CeO2, has been used in catalytic applications like automotive combustion engines, industrial chemical synthesis and solid oxide fuel cells. Recently, ceria has found new applications in biomedicine due to its interesting catalytic and radical scavenging properties, and its low toxicity. The hydroxylated cerium (IV) ions form a reddish orange complex with hydrogen peroxide. This interesting property first discovered by de Boisbaudran was used as the most sensitive test for cerium ions but it has not been utilized as a test for the analysis of other compounds. Later studies have shown that this reaction has two stages: (1) oxidation of Ce(III) to Ce (IV), and (2) complexation of Ce(IV) ion with two molecules of hydrogen peroxide. Ceria nanoparticles are comprised of cerium oxides in mixed valence states both as Ce(III) and Ce(IV) with lower size particles having higher percentage of Ce(III) valence states. This invention is the first to: (1) use ceria nanoparticles as chromogenic indicators in an enzyme assay; (2) immobilize ceria onto a solid support such as paper; and (3) integrate this concept to construct a paper bioassay for the detection of glucose for point-of-care (“POC”) diagnostics.
An electrochemical hydrogen peroxide sensing system is disclosed in Patent No. US 2009/0071848 entitled: “Cerium oxide nanoparticle regenerative radical sensor” by Seal et al. (Seal, Cho et al. 2006). Electrochemical detection of hydrogen peroxide is also described in two research papers (Ispas, Njagi et al. 2008) (Mehta, Patil et al. 2007). In these systems, ceria was used as electrode coating and the signal was obtained by electrochemical means. Such system can be used only in combination with an electrochemical transducer and involves the use of reference/counter electrodes and specialized electrochemical instrumentation (e.g. potentiostat). The sensor is limited to the detection of hydrogen peroxide and superoxide radicals. It is also used in conjunction with platinum, which is an expensive catalyst for hydrogen peroxide by itself. In addition, that sensor might be prone to interferences from other electrochemically active species which can be oxidized or reduced at the applied potential such as hydrogen peroxide.
The relevant art is described in further detail in the following references, all of which are hereby incorporated by reference: Babko, A. K. and A. I. Volkova (1954), “The colored peroxide complex of cerium,” Ukrains'kii Khemichnii Zhurna 20: 211-215; Beach, E. F. and J. J. Turner (1958), “An enzymatic method for glucose determination in body fluid,” Clinical Chemistry 4(6); 462-475; Dungchai, W., O. Chailapakul, et al. (2010), “Use of multiple colorimetric indicators for paper-based microfluidic devices.” Analytica Chimica Acta 674(2): 227-233; Ispas, C., J. Njagi, et al. (2008), “Electrochemical studies of ceria as electrode material for sensing and biosensing applications,” Journal of the Electrochemical Society 155(8): F169-F176; Martinez, A., S. Phillips, et al. (2007), “Patterned Paper as a Platform for Inexpensive, Low-Volume, Portable Bioassays,” Angewandte Chemie International Edition 46(8): 1318-1320; Mehta, A., S. Patil, et al. (2007), “A novel multivalent nanomaterial based hydrogen peroxide sensor,” Sensors and Actuators a-Physical 134(1): 146-151; Seal, S., H. Cho, et al. (2006), “Cerium oxide nanoparticle regenerative free radical sensor,” USA, University of Central Florida, USA. WO 2006130473, US 20090071848: 19 pp; Trinder, P. (1969), “Determination of blood glucose using 4-amino phenazone as oxygen acceptor,” Journal of Clinical Pathology 22(2): 246; Wolfgang, G., U. B. Hans, et al. (1973), “Reagent composition and process for the determination of glucose,” Germany, Boehringer, Mannheim GmbH, U.S. Pat. No. 3,721,607; and Yu, P., S. A. Hayes, et al. (2006), “The phase stability of cerium species in aqueous systems—II. The Ce(III/IV)-H2O-H2O2/O-2 systems. Equilibrium considerations and pourbaix diagram calculations.” Journal of the Electrochemical Society 153(1): C74-C79.